Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential sensitivity to metabolic inhibition

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

Synthesis of deuterated γ-linolenic acid and application for biological studies: metabolic tuning and Raman imaging

γ-Linolenic acid (GLA) is reported to show tumor-selective cytotoxicity via unidentified mechanisms. We introduced deuterium into GLA as a dual functional tag for metabolic inhibition and Raman imaging and applied for mechanistic studies.

CD4+ T cell activation and concomitant mTOR metabolic inhibition can ablate microbiota-specific memory cells and prevent colitis

Microbiota-reactive CD4+ T memory (TM) cells are generated during intestinal infections and inflammation, and can revert to pathogenic CD4+ T effector (TE) cells, resulting in chronicity of inflammatory bowel disease (IBD). Unlike TE cells, TM cells have a low rate of metabolism unless they are activated by reencountering cognate antigen. Here, we show that the combination of cell activation and metabolic checkpoint inhibition (CAMCI), by targeting key metabolic regulators mTORC and AMPK, resulted in cell death and anergy, but enhanced the induction of the regulatory subset. Parenteral application of this treatment with a synthetic peptide containing multiple flagellin T cell epitopes (MEP1) and metabolic inhibition successfully prevented the development of CD4+ T cell–driven colitis. Microbiota-specific CD4+ T cells, especially the pathogenic TE subsets, were decreased 10-fold in the intestinal lamina propria. Furthermore, using the CAMCI strategy, we were able to prevent antigen-specific TM cell formation upon initial antigen encounter, and ablate existing TM cells upon reactivation in mice, leading to an altered transcriptome in the remaining CD4+ T cells after ablation. Microbiota flagellin–specific CD4+ T cells from patients with Crohn’s disease were ablated in a similar manner after CAMCI in vitro, with half of the antigen-specific T cells undergoing cell death. These results indicate that parenteral activation of microbiota-specific CD4+ T cells with concomitant metabolic inhibition is an effective way to ablate pathogenic CD4+ TM cells and to induce T regulatory (Treg) cells that provide antigen-specific and bystander suppression, supporting a potential immunotherapy to prevent or ameliorate IBD.

Ability of chlorhexidine, octenidine, polyhexanide and chloroxylenol to inhibit metabolism of biofilm-forming clinical multidrug-resistant organisms

Purpose: This in vitro study was designed to determine if standard antiseptics used for skin and environmental surface cleansing can disrupt the metabolic activity (as a measure of viability) of multidrug-resistant gram-negative bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus isolates within their native biofilms. Methods: Sixty clinical isolates of multidrug-resistant bacteria were selected for testing in different chlorhexidine gluconate, octenidine, polyhexanide and chloroxylenol concentrations. Metabolic inhibition of biofilm for each clinical isolate was analysed using a biofilm viability assay. Results: Chlorhexidine gluconate (mean = 83.8% ± 9.8%) and octenidine (mean = 84.5% ± 6.8%) showed the greatest efficacy against biofilms of the tested microorganisms, with the greatest efficacies against MRSA. The antiseptics demonstrated the least efficacy against biofilms of Pseudomonas aeruginosa. Conclusion: Chlorhexidine gluconate and octenidine showed the greatest level of bacterial metabolic inhibition and were statistically equivalent. Polyhexanide was more effective than chloroxylenol, but both were inferior to chlorhexidine gluconate and octenidine against the tested organisms.